本文選題：骨髓間充質(zhì)干細(xì)胞 + 胰腺　； 參考：《昆明醫(yī)科大學(xué)》2014年碩士論文

【摘要】：目的 分離、體外培養(yǎng)、鑒定兔的骨髓間充質(zhì)干細(xì)胞(MSCs),建立兔胰、腎聯(lián)合移植(SPKT)小動(dòng)物模型,探討MSC能否誘導(dǎo)胰腎聯(lián)合移植免疫耐受。 材料和方法 1.采用健康幼齡日本大耳白兔,麻醉致死后沖洗骨髓收集骨髓液,采用直接貼壁法分離培養(yǎng)MSCs,對(duì)MSCs進(jìn)行形態(tài)學(xué)觀察,同時(shí)通過流式細(xì)胞分析術(shù)對(duì)CD34、CD44.CD45.CD904種表面抗原進(jìn)行鑒定。取第5代MSCs,加入50μmol/L抗壞血酸鹽2磷酸鹽、50μmol/L消炎痛0.5μmol/L地塞米松誘導(dǎo)其分化。 2.選用日本大耳兔作SPKT的供、受體,切除受體雙側(cè)腎臟,供體腎臟原位吻合,供體胰腺吻合于另一側(cè)；用袖套法結(jié)扎完成供體門靜脈、腎靜脈與受體腎靜脈斷端的吻合；用縫合法完成動(dòng)脈吻合,輸尿管和胰管置管行膀胱內(nèi)引流。 3.術(shù)后存活的移植兔分組(A.B.C.D每組3只),分別給予不同處理,A組：受體胰腎移植后予MSC靜脈輸注,B組：受體胰腎移植后予MSC靜脈輸注+免疫抑制劑(他克莫司膠囊),C組：受體胰腎移植后只給免疫抑制劑(他克莫司膠囊),D組：受體胰腎移植后不行干預(yù)；取各組移植兔術(shù)前和術(shù)后第1、3天共3個(gè)時(shí)間段靜脈血,應(yīng)用流式細(xì)胞儀檢測(cè)CD4+T細(xì)胞和CD8+T細(xì)胞百分率,并計(jì)算其比值及CD4+CD25+Treg細(xì)胞的比例。 結(jié)果 1.第2代MSCs經(jīng)流式細(xì)胞技術(shù)鑒定出細(xì)胞的CD34(-).CD45(-).CD44(+). CD90(+)符合MSCs的表面抗原,第5代骨髓間充質(zhì)干細(xì)胞,成脂誘導(dǎo)10d后油紅O染色顯示有大量脂質(zhì)沉積并相互融合,細(xì)胞由長梭形變?yōu)槎噙呅?證明了干細(xì)胞的分化特性,培養(yǎng)的細(xì)胞就是骨髓間充質(zhì)干細(xì)胞。 2.15只供體順利完成胰腺和腎臟切取術(shù)：15只受體順利完成胰腎聯(lián)合移植術(shù)；供體腎臟、胰腺移植后迅速恢復(fù)血液循環(huán),無出血及漏血,術(shù)后3只死于持續(xù)性低血壓,實(shí)驗(yàn)組的3只受體術(shù)后平均生存期(4.0±0.7)d；對(duì)照組的9只受體術(shù)后平均生存期(4.0±0.5)d,成功建立兔胰腎聯(lián)合移植模型。 3.術(shù)后第1、3天輸注MSC組(n=3)比未干預(yù)組(n=3)CD4+T細(xì)胞數(shù)和CD4+CD25+Treg細(xì)胞數(shù)比例高,差異有統(tǒng)計(jì)學(xué)意義(p0.05)。 4.術(shù)后第1、3天輸注MSC組(n=3)比輸注免疫抑制劑組(n=3)CD4+T細(xì)胞數(shù)和CD4+CD25+Treg細(xì)胞數(shù)比例低,差異有統(tǒng)計(jì)學(xué)意義(p0.05)。 5.術(shù)后第1、3天輸注MSC+免疫抑制劑組(n=3)比輸注免疫抑制劑(n=3)CD4+T細(xì)胞數(shù)和CD4+CD25+Treg細(xì)胞數(shù)比例高,差異有統(tǒng)計(jì)學(xué)意義(p0.05)。 結(jié)論 MSC能調(diào)節(jié)移植胰、腎免疫反應(yīng),減輕排斥反應(yīng),但效果沒有免疫抑制劑明顯,不能替代免疫抑制劑。
[Abstract]:Objective to isolate and culture rabbit bone marrow mesenchymal stem cells (MSCs) and to establish a small animal model of pancreas and kidney transplantation (SPKT) in order to investigate whether MSC can induce the immune tolerance of simultaneous pancreaticorenal transplantation. Materials and methods 1. Bone marrow fluid was collected from the bone marrow of young Japanese white rabbits after anaesthesia. MSCs were isolated and cultured by direct adherent method. The morphology of MSCs was observed, and CD34, CD44.CD45.CD904 surface antigens were identified by flow cytometry. The fifth generation of MSCs was induced by 50 渭 mol / L ascorbate 2 phosphate 50 渭 mol / L indomethacin 0.5 渭 mol / L dexamethasone. Japanese big ear rabbits were used as donor and recipient of SPKT, bilateral kidney was resected, donor kidney was anastomosed in situ, donor pancreas was anastomosed on the other side, portal vein of donor was ligated with cuff method, anastomosis of renal vein and renal vein of recipient was completed. Arterial anastomosis was performed by suture and intravesical drainage of ureter and pancreatic duct was performed. The surviving rabbits were divided into three groups of A.B.C.D in each group and treated with different treatments: group A: group B: after transplantation of recipient pancreas and kidney, immunosuppressive agent MSC (tacrolimus capsule) was given. Group D: only immunosuppressive agents were given to recipients after pancreaticorenal transplantation (tacrolimus capsule group D: no intervention after recipient pancreaticorenal transplantation; The percentage of CD4 T cells and CD8 T cells were measured by flow cytometry, and the ratio of CD4 CD25 Treg cells were calculated. Result 1. The second passage of MSCs was identified by flow cytometry. CD90 () conformed to the surface antigen of MSCs. After 10 days of adipogenic induction, oil red O staining showed a large amount of lipid deposition and fusion, and the cells changed from fusiform to polygon, which proved the differentiation of stem cells. The cultured cells were bone marrow-derived mesenchymal stem cells. 2.15 donors successfully completed pancreas and kidney excision, 15 recipients successfully completed pancreaticorenal transplantation, donor kidneys, pancreas transplantation, and rapid recovery of blood circulation. Without bleeding or bleeding, 3 died of persistent hypotension after operation. The mean survival time of 3 recipients in the experimental group was 4.0 鹵0.7 days, and the average survival time of 9 recipients in the control group was 4.0 鹵0.5 days. The model of simultaneous pancreaticorenal transplantation was successfully established in rabbits. The numbers of CD4 T cells and CD4 CD25 Treg cells in MSC group were higher than those in control group on the 1st day after operation, and the difference was statistically significant (P 0.05). The number of CD4 T cells and CD4 CD25 Treg cells in MSC group was lower than that in immunosuppressive group on the 1st day after operation (P < 0.05). The ratio of CD4 T cells and CD4 CD25 Treg cells in MSC immunosuppressant group was significantly higher than that in MSC immunosuppressant group on the 1st day after operation (P 0.05). Conclusion MSC can regulate the immune response of pancreas and kidney and alleviate the rejection, but the effect is less than that of immunosuppressant and can not replace the immunosuppressant.
【學(xué)位授予單位】：昆明醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2014
【分類號(hào)】：R657.5;R699.2

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